Epstein-Barr virus (EBV) infected cells were examined in three cases of EBV-associated hemophagocytic syndrome (EBV-AHS) by analysis of the heterogeneity of terminal repetitive sequences in the EBV genome, indicating monoclonal expansion of EBV-infected cells in all cases. Involvement of T lymphoid cells was determined by the finding of in situ hybridization using [35S]-labeled RNA probes specific for the small EBV-encoded nuclear RNAs, EBER1 and EBER2, in combination with immunostaining for the TCR-beta chain, CD45RO, CD20, CD30 and CD68 antigens in these three cases. The majority of lymphoid cells showing EBER transcripts were stained by antibodies against CD45RO and TCR-beta. In contrast, EBER-specific signals were not detectable on B cells or hemophagocytic cells. These data support the concept that subclinical EBV-associated T cell proliferation is the primary characteristic of EBV-AHS, rather than proliferations of hemophagocytosing histiocytes.